Nature uses an amazing array of enzymes to make small molecule natural products. Among the most interesting but least understood enzymes making these compounds are the iterative polyketide synthases (IPKSs) found in filamentous fungi. In contrast to the well-studied bacterial type I PKSs that operate in an assembly-line fashion, IPKSs are megasynthases that function iteratively by using a single set of catalytic domains repeatedly in different combinations to produce structurally diverse fungal metabolites. Bioinformatics analysis of the genomes of recently sequenced fungal species revealed that each genome contains a large number of genes encoding IPKSs. The total numbers of IPKSs significantly outnumber the known polyketides and polyketide-nonribosomal peptides isolated from these species, suggesting that a majority of biosynthetic genes are silent in these fungi under cultivating conditions. This in turn suggests that the fungal species may have untapped potential to synthesize a much large number of natural products. Furthermore, analysis and engineering of IPKSs have been hampered by inability to obtain sufficient amounts of the functional purified megasynthase from either the native fungal host or heterologous Aspergillus hosts. As a result, the programming that governs metabolite assembly by IPKSs is not understood. Key aspects that remain to be elucidated include: 1) the catalytic and structural roles of each domain in the megasynthase;2) substrate specificities of the catalytic domains and their tolerance to perturbation in megasynthase functions;and 3) factors governing the choice of different combinations of catalytic domains during each iteration of catalysis. The objective of this proposal is to develop the genetically superior Saccharomyces cerevisiae as a heterologous host for reconstitution, analysis and engineering of IPKSs, especially the enigmatic highly-reducing IPKS, such as LovB associated with Lovastatin biosynthesis. We have accumulated a significant body of preliminary data to demonstrate that S. cerevisiae is a highly robust host for expressing these megasynthases in functional forms, and can facilitate the production of polyketide products both in vivo and in vitro with purified enzymes. The following specific aims will be pursued: 1) Engineer and optimize S. cerevisiae towards producing fungal metabolites and megasynthases;2) Reconstitution of fungal megasynthases in S. cerevisiae;3) Biochemical analysis of fungal PKS using S. cerevisiae;and 4) Genome mining of filamentous fungi using S. cerevisiae as a host. PUBLIC HEALTH RELEVANCE: Filamentous fungi are a rich source of natural products. Among them, polyketides represent an important family of structurally diverse natural products. Polyketides are produced by polyketide synthase (PKSs). Genome sequencing of many fungal species has revealed each organism contains a large number of PKSs, yet many of these PKSs have unknown functions or produce unknown metabolites. Therefore, filamentous fungi can be considered "underachievers" of natural product producers. We propose here to use Saccharomyces cerevisiae as an expression host to heterologously produce fungal PKSs. We will use this genetically superior host to reconstitute fungal PKSs of both known and unknown functions, perform genome mining of sequenced fungal species and engineered biosynthesis of new fungal-derived natural products.